Identification of a functional glucocorticoid response element in the CYP3A1/IGC2 gene.
The rat CYP3A subfamily of cytochrome P450 consists of steroid- and drug-metabolizing enzymes inducible by pregnenolone 16alpha-carbonitrile and by supra-physiological doses of dexamethasone. The induction of CYP3A by dexamethasone has been proposed to be mediated by a mechanism distinct from the glucocorticoid receptor mediated response. However, a synergistic induction of CYP3A has been observed with physiological doses of glucocorticoids and other CYP3A inducers. We have identified the presence of a glucocorticoid-responsive element in the CYP3A1/IGC2 gene that mediates the induction with physiological doses of glucocorticoids. A 219-bp dexamethasone responsive fragment of the CYP3A1/IGC2 gene localized at -2100/-1882 bp upstream of the transcription initiation site was identified in transfection experiments with HepG2 cells. Maximum induction was achieved with 50-100 nM dexamethasone. DNase I footprinting analysis revealed two glucocorticoid receptor-protected sequences in the 5' flank of the CYP3A1/IGC2 gene. Point mutations in footprint I (-1982/-1960-bp) completely abolished binding and transcription activation whereas a mutation in footprint II (-2001/-1986-bp) only decreased the binding and had no effect on transcription activation. These results led to the conclusion that the glucocorticoid response element present in footprint I mediated the dexamethasone response in transfection experiments with HepG2 cells. Pregnenolone 16alpha-carbonitrile failed to induce any transcriptional effect mediated by this response element in the HepG2 cells.
Pereira
T M
TM
Department of Medical Nutrition, Karolinska Institute, Huddinge University Hospital, Novum, Sweden.
Carlstedt-Duke
J
J
Lechner
M C
MC
Gustafsson
J A
JA
D000818:Animals; D001483:Base Sequence; D051544:Cytochrome P-450 CYP3A; D003577:Cytochrome P-450 Enzyme System / Q000235:genetics*; D018983:DNA Footprinting; D004268:DNA-Binding Proteins / Q000378:metabolism; D005786:Gene Expression Regulation; D005938:Glucocorticoids / Q000502:physiology*; D006801:Humans; D006899:Mixed Function Oxygenases / Q000235:genetics*; D008969:Molecular Sequence Data; D011401:Promoter Regions, Genetic; D051381:Rats; D011965:Receptors, Glucocorticoid / Q000502:physiology; D017384:Sequence Deletion; D014162:Transfection; D014407:Tumor Cells, Cultured
D004268:DNA-Binding Proteins; D005938:Glucocorticoids; D011965:Receptors, Glucocorticoid; D003577:Cytochrome P-450 Enzyme System; D006899:Mixed Function Oxygenases; C104464:CYP3A protein, human; D051544:Cytochrome P-450 CYP3A